The present invention relates to diagnosing and possible treatment of vascular disorders, and, in particular, to providing a highly efficient localizing agent which exhibits high affinity for certain vascular disorders.
The clotting of blood as a part of the body's response to an injury or trauma is a part of the natural phenomenon of hemostasis. The clotting of blood, however, may occur in places in the body which produce results which, at times, can be life threatening. Among the more frequent examples of unwanted clotting occurring in the vascular system are those found in the deep veins of the legs or in the lungs. The blockage of blood by a clot in a blood vessel can stop the supply of blood to vital organ systems and other body parts. Equally life-threatening are clots which become dislodged from their original sites and flow through the circulatory system causing blockages at more remote sites. These dislodged clots are called embolisms.
Blood clot formation derives from a series of events, called the coagulation cascade, in which the final steps involve the formation of the enzyme thrombin. Thrombin converts circulating fibrinogen protein into fibrin which in turn forms a mesh-like structure known as the blood clot. The coagulation cascade, (schematically shown in FIG. 1), is highly regulated and involves a series of transformations caused by a succession of zymogen activations, wherein the activated form of one enzyme factor initiates the activation of the next enzyme. The numerous steps of the cascade may be significantly amplified to assure a rapid response to trauma or injury. The cascade is completed when highly soluble fibrinogen is converted by the proteolytic enzyme thrombin into an insoluble fibrin clot.
Under normal hemostatic conditions, a fibrin clot is eventually dissolved by the process of fibrinolysis. As the damaged blood vessel is repaired, the fibrinolytic enzyme plasmin is activated to dissolve the formed fibrin clot.
The detection of blood clots and the subsequent therapeutic dissolution thereof has long been a profoundly important clinical goal, especially since abnormal or pathological clotting is often life threatening and requires medical intervention. Thus, the need for detection and the ability to safely lyse or dissolve pathological clots is of great importance to the medical community. Some affinity of whole or intact albumin, whether in the macroaggregated or microsphere form, for fibrin is known in the art. Common diagnostic uses of the albumin molecule include combining the albumin molecule with a radio isotope for the purposes of obtaining a scintigraphic image of visceral organs.
Albumin is a type of globular protein and is present in mammalian blood and tissues. Albumin is the most abundant protein in human serum and is thought to perform at least two important physiologic functions: osmotic regulation and transportation of fatty acids between adipose tissues.
Most often, when human serum albumin is used for organ imaging, it is denatured by heating so as to produce albumin aggregates of varying particle size and then selectively sieved to obtain the desired particle size. When used diagnostically, albumin is most often combined with a radioisotope, such as technetium-99m or iodine 131, so that scintigraphic images may be obtained. Human albumin microspheres (HAM) and macroaggregated albumin (MAA), are the most common denatured albumin products used diagnostically and such compounds are often referred to as organ imaging agents. Both human albumin microspheres and macroaggregated albumin are denatured by a heating process wherein the albumin protein aggregates in the presence of heat. The denatured albumin is then suspended in a buffered solution before injecting into the vascular system of the patient. U.S. Pat. No. 3,674,900 to Thompson, U.S. Pat. No. 3,803,299 to Nouel, and U.S. Pat. No. 3,862,299 to Bruno, et al., all disclose the use of macroaggregates of serum albumin labeled with a radio nuclide such as technetium-99m for the purposes of studying pulmonary vascular circulation. An article by Webber, "Albumin Aggregates for Detection of Clots", Seminars in Nuclear Medicine, Volume VII, No. 3 (July), 253-261, (1977), states that macroaggregates of albumin, (MAA), vary in uptake and that MAA particles from different manufacturers and possibly even from the same manufacturer at various times differ in activity.
U.S. Pat. Nos. 4,337,240 and 4,226,846 to Saklad disclose the use of microaggregates of albumin useful for evaluating the reticuloendothelial system including the liver, lymphatic system and bone marrow. Diagnostic techniques employing the above mentioned albumins and radio nuclides have gained widespread acceptance since their introduction and are an important adjunct to obtaining a definitive diagnosis of vascular pathology. Such products, while useful, are often difficult to prepare and limited in their usefulness. The affinity of albumin for fibrin in mammalian vasculature is weak and difficult to show. This problem often limits the usefulness of this diagnostic modality along with albumin based diagnostic agents since the weak binding will often yield studies of indeterminate result as to the underlying pathology of the suspected vascular block. The lack of intensity with which prior art whole albumin aggregates are able to bind to fibrin in the vascular system therefore limits the usefulness of albumin-based products for diagnostic purposes in cases such as for studies to rule out deep venous thrombosis. Currently, the radionuclide-labeled albumin aggregate-based products are often limited to organ scans such as those of the lung or liver. The high number of indeterminate or inconclusive studies resulting from using albumin aggregate-based products, as known in the prior art, for diagnostic purposes for deep venous thrombosis is attributable to the lack of intense binding of the albumin product to the clot. Thus, the use of such products has failed to meet expectations because of the low level of binding by the albumin-based products at the site of the occlusion, especially in the case of deep venous thrombosis.
The unreliability of albumin based products in diagnosing deep venous thrombi has led to the use of radionuclide-labeled fibrinogen products. U.S. Pat. No. 3,933,996 to Charlton, et al. disclose a composition of albumin and radioactive fibrinogen for detecting thrombi in the deep veins of the leg. Fibrinogen, however, is an inherently unstable material, much more so than other common blood proteins even when precautions have been taken.
To date, radio-labeled products of human serum albumin have also suffered from a lack of stability, an inability to withstand significant changes in pH, and short effective useful lives. Further, difficulties in the heating and controlling of the coagulation of the albumin have lead to difficulty in obtaining economical yields of the particular particle sizes which are generally within the range of 0.1 to 5 microns for microaggregated human serum albumin. On the other hand, macroaggregated human serum albumin, having a particle size of greater than 5 microns but less than 100 microns, may exhibit difficulties in dissolving after introduction into mammalian vasculature due to their particle size.
Kowalsky, et al. in Radio Pharmaceuticals in Nuclear Medicine Practice, at page 239, describes the difficulties encountered in the production of albumin microspheres. A small volume of aqueous albumin solution is mixed in a large volume of vegetable oil to produce an emulsion of minute spheres disbursed within the oil. Sphere size is dependent upon mixing speed and the ratio of albumin to the oil. Spheres are then dried and sieved to obtain appropriate particle size. Spheres are then treated with a stannous chloride solution and a radionuclide such as technetium-99m pertechnetate before injection into the vascular system of the subject for study. Other problems include irregularities in particle size, particle number, particle hardness and chemical composition. Further, biodegradation of macroaggregated albumin or human albumin microspheres particles and their radio labels must be regulated so as to provide an ample time for imaging on the one hand, yet once imaging is completed, a somewhat rapid degradation and elimination from the body on the other.
Unlike coagulated, denatured albumin products, albumin which has been cleaved into fragments has not been used in vivo for the purposes of diagnosing vascular system pathology in mammals. The uses for these fragments of albumin have been limited to the study of the albumin molecule structure and in U.S. Pat. No. 1,371,380 to Kottmann, which discloses the preparation of compounds which contain combinations of metals with albumins. Example 13 of the '380 Patent to Kottmann describes the preparation of a compound from iron chloride and placenta albumin which has been treated with trypsin. The compound was then used in vitro as a purported pregnancy test.
The aforementioned approaches and techniques for employing albumin based diagnostic agents all fall short of providing a diagnostic agent which possesses the ability to exhibit a higher percentage of binding of the albumin to the fibrin based pathology in the vascular system of mammals. Further, the prior art fails to provide a reliable albumin based product which is useful in the diagnosis of deep venous thrombosis and is capable of minimizing the number of diagnostic tests for the presence of thrombosis having an indeterminative result.
Accordingly, it is an object of the present invention to provide an effective albumin fragment based diagnostic product detects fibrin found in the vascular system of mammals.
It is also an object of the present invention to provide an agent capable of detecting thrombosis and embolism in patients which is free of the disadvantages of the prior art preparations noted above.
It is a further object of the present invention to provide an albumin fragment based diagnostic product which possesses anticoagulant, anti-fibrinolytic and low clot retraction properties.
A still further object of the present invention is to provide a diagnostic agent which demonstrates a tighter binding to fibrin than is available in the prior art.
Yet another object of the present invention is to provide an efficient high-affinity agent for binding to fibrin along with the advantages concomitant with such high affinity agent such as advancement in treatment of vascular disorders.
Other objects and advantages will become apparent to those skilled in the art from a consideration of the ensuing description which proceeds with reference to the following illustrative claims.